Hidejiro Kawahara

Dendritic Cells Fused with Allogeneic Colorectal Cancer Cell Line Present Multiple Colorectal Cancer–Specific Antigens and Induce Antitumor Immunity against Autologous Tumor Cells

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2−/HLA-24−), carcinoembryonic antigen (CEA)+, and MUC1+ as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2+ and/or HLA-A24+) and allogeneic COLM-6 resulted in MHC class I– and MHC class II–restricted proliferation of CD4+ and CD8+ T cells, high levels of IFN-γ production in both CD4+ and CD8+ T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells.

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor‐associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte‐derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA‐derived CEA and MUC1 antigens and DC‐derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4+ and CD8+ T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN‐γ. More importantly, coculture of fusion cells with patient‐derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen‐specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1‐positive and HLA partially matched target cells. Antigen‐specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA‐ and MUC1‐specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen‐specific CTL responses. Our report demonstrates the simultaneous induction of CRCA‐specific CTL responses restricted by HLA‐A2 and ‐A24.

Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- γ at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+IFN-γ+CD8+ T cells and CD154+ IFN-γ+CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.

The herbal medicine Dai-Kenchu-To directly stimulates colonic motility

Dai-kenchu-to (DKT) has attracted attention as a drug that improves the symptoms of postoperative ileus. However, the detailed mechanism of its action still remains unknown. The effect of DKT on colonic motility was herein evaluated using an original method. Eight healthy male volunteers who understood the purpose of this study were enrolled. Dai-kenchu-to (5 g) was dissolved in saline and administered into the cecum using a colonoscope until the ascending colon became distended. Colonic motility was observed by extrasomatic ultrasonography for 30 min. Colonic contractions were observed 129.4 (range 110–145) s after DKT administration into the ascending colon. Every segment in the right colon divided by the crescentic folds contracted independently. On the other hand, no colonic contractions were observed in the right colon after saline solution alone was administered to the ascending colon. In conclusion, DKT stimulates colonic motility immediately after administration, in the same manner as it does for the upper alimentary tract.

Umbilical Incision Laparoscopic Surgery with One Assist Port for Anterior Resection

Background: A high surgical technique is required for laparoscopic anterior resection using single-incision laparoscopic surgery such as multiport surgery. We report a novel surgical technique of umbilical incision laparoscopic surgery with one assist port which could be performed like conventional multiport surgery. Methods: With the patient in the lithotomy position, a 3-cm longitudinal skin incision is made at the umbilicus and carried down to the peritoneum. A 12-mm and two 5-mm trocars are placed through the incision. Another 12-mm trocar is placed for the insertion of laparoscopic coagulation shears and a stapler in the right lower quadrant. After surgery, the trocar in the right lower quadrant is removed and a drain is replaced through the insertion site. The operator mainly uses two trocars, a 5-mm trocar placed at the umbilicus and a 12-mm trocar in the right lower quadrant, making it like conventional laparoscopic surgery. Results: The median operation time was 195 (range 180–205) min, intraoperative blood loss was 20 (range 0–60) ml, and none of the 3 patients had any complications postoperatively. Conclusion: As opposed to surgery with only three ports, so-called single-incision laparoscopic surgery, this operation can be performed much more comfortably if another port is inserted in the right lower quadrant.

New Approach for Laparoscopic Surgery of the Right Colon

Background: In patients in whom the ventral aspect of the root of the mesentery was obscured by the adherent greater omentum, laparoscopic surgery is usually abandoned. To forcefully loosen the adhesions by a laparoscopic maneuver may cause inadvertent intestinal injury. We describe a procedure, named the ‘retromesen teric approach’, which enables a safe laparoscopic right-sided colic operation in such circumstances. Patients and Methods: From 2000 to 2003 in our institute, a laparoscopic right-sided colic operation was performed in 21 patients in whom the ventral aspect of the root of the mesentery was obscured by the adherent greater omentum using the retromesenteric approach (RMA; n = 16) and a conventional approach (CA; n = 5). We reviewed the medical records for the operative duration, intraoperative blood loss, conversion rates and postoperative complications. Results: The duration of operation in the RMA group ranged from 75 to 120 (median 95) min, which was shorter than that in the CA group (p < 0.05). Perioperative bleeding in the RMA group ranged from 0 to 115 (median 30) ml, which was smaller than that in the CA group (p < 0.05). No conversion and no postoperative complications were noted in the both groups. Conclusions: Our new technique, in which the right colon and the ileum are dissected and lifted en bloc from the retroperitoneum, is safe and useful for laparoscopic right-sided colic operation.

Where Does the First Lateral Pelvic Lymph Node Receive Drainage from

Background: Lateral pelvic lymph node dissection (LPLD) in the treatment of rectal cancer has risks and benefits. Avoidance of unnecessary LPLD is important, however, preoperative and/or intraoperative accurate detection of lateral lymph node metastases have not been established. If the lateral lymph node to which the fluid first spread from the primary lower rectal cancer is detected accurately, it may guide the need for LPLD and may assist in avoiding unnecessary dissection. Methods: A total of 14 patients with T3 lower rectal cancer were evaluated to locate the lymph nodes through which indocyanine green (ICG) reached the lymphatics. After ICG was injected into the lower rectum via an endoscope preoperatively, total mesorectal excision was first performed, and LPLD was performed with infrared ray electronic endoscopy (IREE) to assess the degree of retention of ICG in each regional lymph node. Results: Drainage of ICG to lateral pelvic lymph nodes was observed in 6 of 14 patients (43%). All ICG-containing lymph nodes were detected by IREE. When present, lateral pelvic wall lymph node drainage was limited exclusively to the peri-internal iliac artery nodes. No obturator nodes were involved. Conclusion: The first lateral lymph node that receives lymphatic drainage from lower rectal cancer is located around the internal iliac arteries.

Lateral pelvic lymph node dissection using latero-vesical approach with aspiration procedure for advanced lower rectal cancer.

BACKGROUND/AIMS The aim of this study was to evaluate the impact of complete dissection of areolar tissue surrounding lymph nodes in lateral pelvic lymphadenectomy on the outcome of advanced rectal cancer at or below the peritoneal reflection. METHODOLOGY From 1995 to 2004, lateral pelvic lymph node dissection was performed in 141 consecutive patients with advanced rectal cancer at or below the peritoneal reflection by open surgery in our hospital. They were divided into two groups according to the techniques used for lymph node dissection, i.e. conventional method (CM) and our original method, latero-vesical approach with aspiration procedure (LVA), which eliminates not only lymph nodes but also the tissue surrounding the lymph nodes. RESULTS The number of dissected lateral pelvic lymph nodes by LVA was significantly higher than that by CM. In patients without lateral pelvic lymph node metastasis, no significant difference in the outcome was observed between the two groups. On the contrary, among the patients with lateral pelvic lymph node metastasis, five-year survival rates of the group with CM or with LVA was 50% and 70% respectively. CONCLUSIONS For patients with lateral pelvic lymph node metastasis, lateral pelvic lymphadenectomy, complete dissection of areolar tissue surrounding lymph nodes, may contribute to improve the prognosis of advanced rectal cancer at, or below, the peritoneal reflection.

Appendiceal Mucocele Detected under Treatment of Ulcerative Colitis

A 33-year-old female patient with ulcerative colitis was referred to our outpatient clinic in January 2008 with right lower abdominal pain without bloody diarrhea. Colonoscopy found mild proctosigmoiditis and a submucoal tumor with a maximal diameter of 5 cm in the cecum. Computed tomography revealed a large, hypodense, cystic cylindrical structure extending to the pelvic space. For severe pain, she underwent partial resection of the cecum including the tumor in March 2008. Intraoperatively, the vermiform appendix was swollen like a sausage and compressing the cecum, which accounted for what appeared to be a submucosal tumor like a volcano by endoscopy. Lymphadenectomy was not performed because malignancy was not suspected. In the surgical specimen, the vermiform appendix was spindle-shaped and contained a large quantity of viscous liquid. Postoperative pathological diagnosis was mucinous cystadenoma, and no cancer cells were present in the viscous liquid within the vermiform appendix. The patient left the hospital 7 days postoperatively, and her colitis remains in remission without any complications.